MODULATION OF OVULATION FIELD OF THE INVENTION
The present invention relates the identification of a novel domain of functional significance on GDF-9 and GDF-9B molecules and to agonists and antagonists which interact therewith to modulate the biological activity of these molecules to alter mammalian ovarian function and ovulation rate.
BACKGROUND OF THE INVENTION
GDF-9 and GDF-9B (also known as BMP 15) are expressed in the oocyte of the developing follicle and play a role in mammalian fertility (Fitzpatrick et al,1998). GDF9 is a member of the transforming growth factor beta (TGFJ3) superfamily (McPherron and Lee, 1993) which is expressed in oocytes from the primary stage of follicular development until ovulation (McGrath et aL, 1995; Laitinen et aL, 1998). GDF9B is closely related to GDF9 (Dube et alt 1998; Laitinen et aL, 1998) and is expressed in mouse oocytes at the same time as GDF9, but in human primary follicles slightly later than GDF9. In the ovary GDF9 and GDF9B have now been shown to be expressed in the developing oocyte in humans (Aaltonen et aL, 1999), rodents (Laitinen et aL, 1998; Dube et aL, 1998; Jaatinen et aL, 1999), ruminants (Bodensteiner et al, 1999; Bodensteiner et aL, 2000; Galloway et aL, 2000) and marsupials (Eckery et aL, 2002). In sheep expression of GDF9 can be seen in primordial follicles .whereas GDF9B is expressed in primary follicles (Bodensteiner et aL, 1999; Galloway et aL, 2000).
GDF9 and GDF9B, like most other members of the TGFf} family, are coded as prepropeptides containing a signal peptide, a proregion and a C-terminal mature region which is the biologically active peptide. Cleavage of the mature region from the proregion is carried out by an intracellular furin-like protease, and occurs at a conserved furin protease cleavage site. Most members of the TGF(3 superfamily are biologically active as dimers, and although GDF9 and GDF9B do not contain the cysteine molecule responsible for covalent interchain disulphide bonding seen in nearly all members of die family, these molecules are thought to be biologically active as dimers (Galloway et aL, 2000; Yan et aL, 2001). However it is unclear whether the physiologically active dimers are homodimers (GDF9-GDF9 and

GDF9B-GDF9B), or heterodimers (GDF9-GDF9B) or whether all three dimer forms play a role. It has been postulated based on the above models that GDF9 homodimers play a more important role in the mouse but in sheep the GDF9B homodimers are the most bioactive (Yan et aL, 2001). It is unclear whether any such difference is related to the fact that sheep are mono-ovulatory animals (maturing usually only one egg per cycle) whereas mice are poly-ovulatory. Clearly both GDF9 and GDF9B play crucial roles in controlling and maintaining fertility in mammals, and understanding the nature of their actions is essential for the development of therapies.
Jeffery et al., 2003 , used a bioinformatics tool (GoCore) to analyse conserved regions across a number of different TGF-p family members and for GDF-9 and GDF-9B across different species, in an attempt to identify regions of functional significance. However, this study did not correlate regions identified as possible functionally significant sites with effects on modulation of ovulation in vivo.
It is an object of the present invention to identify domains of functional significance on the GDF-9 and/or GDF-9B molecules and to identify agonists and antagonists which interact with such domains or mimic such domains to modulate their biological activity thereby modulating mammalian ovarian function and ovulation rates in vivo; and/or to provide the public with a useful choice.
The term "comprising" as used in this specification and claims means "consisting at least in part of, that is to say when interpreting independent claims including that term, the features prefaced by that term in each claim all need to be present but other features can also be present.
SUMMARY OF THE INVENTION
The present invention is based on the identification of a novel domain of functional significance on the GDF-9 and GDF-9B molecules and to agonists and antagonists that interact therewith.

More specifically, the present invention is based on the identification and characterisation of a domain of functional significance around the N-terminal end of the mature GDF-9 and GDF-9B molecules, wherein the N-terminal domain of GDF-9 comprises the amino acid sequence:
DQESASSELKKPLVPASVNLSEYFKQFLFPQNEC (SEQ ID NO: 1); and
the N-terminal domain of GDF-9B comprises the amino acid sequence:
QAGSIASEVPGPSREHDGPESNQC (SEQ ID NO: 2).
The present invention is thus directed to an isolated fragment of the GDF-9 N-terminal
domain, having the ability to modulate ovulation in a female mammal, wherein said GDF-9
N-terminal domain consists of the amino acid sequence
DQESASSELKKPLVPASVNLSEYFKQFLFPQNEC (SEQ ID NO: 1), or a functional derivative, homolog, analog or mimetic thereof
Preferably, the peptide fragment comprises at least one amino acid from positions 1-9 or 25-34ofSEQ ID NO: 1.
The present invention is also directed to an isolated peptide fragment of the GDF-9B N-terminal domain, having the ability to modulate ovulation in a female mammal, wherein said GDF-9B N-terminal domain consists of the amino acid sequence QAGSIASEVPGPSREHDGPESNQC (SEQ ID NO: 2), or a functional derivative, homolog, analog or mimetic thereof.
Preferably the peptide fragment comprises at least one amino acid from positions 1-5 or 21-23 ofSEQIDNO:2.
The amino acid symbols correspond to the one letter code as set out in Table 1, below.
The isolated peptide fragments of the invention preferably comprise at least 5 contiguous amino acids of SEQ ID NO: 1 or SEQ ID NO: 2, or a functional derivative, homolog, analog or mimetic thereof. More preferably, the peptide fragments comprise at least 8, at least 10, at least

12, at least 14, at least 16, at least 18 or at least 20 contiguous amino acids of SEQ ED NO: 1 or SEQ ID NO: 2, or a functional derivative, homolog, analog or mimetic thereof.
Most preferably the peptide fragments of the invention are selected from the group comprising:
DQESASSELKKPLV(C) (SEQ ID NO: 3);
SEYFKQFLFPQNEC (SEQ ID NO: 4);
QAGSIASEVPGPSR(C) (SEQ ED NO: 5); and
SREHDGPESNQC (SEQ ID NO: 6);
or a functional derivative, homolog, analog or mimetic thereof.
Amino acids in parentheses refer to amino acids which are optionally added for conjugation purposes.
The present invention is also directed to an antibody or antibody fragment that binds to one or more of the peptide fragments of the invention.
In a further aspect the present invention provides a method of modulating the ovulation rate of a female mammal, said method comprising the step of administering to said mammal an effective amount of one or more isolated peptide fragments and/or antibodies or antibody fragments of the invention that are capable of interacting with the N-terminal domain of GDF-9 and/or GDF-9B as defined above, and altering the biological activity thereof.
Preferably, the invention provides a method of modulating the ovulation rate of a female mammal, comprising administering to said mammal an effective amount of one or more peptides selected from SEQ ID NOs 3-6, or a functional variant thereof, and/or an antibody or antibody fragment that binds thereto.
In a further aspect the present invention provides a use of one or more isolated peptide fragments of the invention and/or an antibody or antibody fragment that binds thereto, in the manufacture of a medicament for modulating the ovulation rate of a female mammal-


fragment that binds thereto, in the manufacture of a medicament of modulating the ovulation rate of a female mammal.
In a still further aspect, the present invention provides a pharmaceutical composition comprising one or more isolated peptide fragments of the invention that are capable of interacting with the N-terminal domain of GDF-9 and/or GDF-9B defined above, together with a phannaceutically acceptable carrier or excipient
Preferably, the composition comprises one or more peptides selected from the group comprising SEQ ID NOs 3-6 or a functional variant thereof, together with a phannaceutically acceptable carrier or excipient The composition may additionally or alternatively comprise an antibody or antibody fragment that binds to one or more of said peptides, together with a phannaceutically acceptable carrier or excipient.
DESCRIPTION OF THE DRAWINGS
The invention will now be described in more detail with reference to the figures of the
accompanying drawings in which:
Figure 1 shows the position of peptides of SEQ ID NOs 3 and 4 on GDF-9;
Figure 2 shows the position of peptides of SEQ ID NOs 5 and 6 on GDF-9B;
Figures 3a and 3b show the sequence homology of GDF9 and GDF-9B from a number of
different species and the N-terminal domain consensus Sequences;
Figure 4 shows the effect of treatment with GDF-9 and GDF-9B peptides on antral follicle
development in bovine. (A - control, keyhole limpet haemocyanin (KLH) alone; B = KLH-
GDF-9 (SEQ ID NO: 7); C = KLH-GDF-9B (SEQ ID NO: 5); D = KLH-GDF-9 and KLH-GDF-
9B (SEQ ID NOS: 5 and 7);
Figure 5 shows the effect of a polyclonal antibody from sheep immunized against ovine GDF-9
peptide SEQ ID NO 3 or SEQ ID NO 4, or GDF-9B peptide SEQ ID NO 5 on ovine (o) GDF-9
and oGDF-9B stimulated 3H-thymidine incorporation of rat granulosa cells when added directly
to the bioassay. The horizontal line indicates the level of response of the granulosa cells treated
with control media (i.e. media without oGDF-9 or oGDF-9B). Data presented are the mean and
standard error of the mean of 4 replicate experiments. *P<0.05, **P<0.01 versus control media
treated cells, ^p<0.05, ''PO.Ol versus oGDF-9+oGDF-9B treated cells that did not receive
antibodies;

Figure 6 shows the effect of a polyclonal antibody from sheep immunized against ovine GDF-9 peptide SEQ ID NO 4 on marine (m) GDF-9 and ovine (o) GDF-9B stimulated 3H-thymidine incorporation of rat granulosa cells when added directly to the bioassay. The horizontal line indicates the level of response of the granulosa cells treated with control media (i.e. media without mGDF-9 or oGDF-9B). Data presented are the mean and standard error of the mean of 3 replicate experiments. *P<0.053 **P<0.01 versus control media treated cells, P≤O.Ol versus mGDF-9+oGDF-9B treated cells that did not receive antibodies;
Figure 7 shows the effect of differing doses of a polyclonal antibody from sheep immunized against ovine GDF-9 peptide SEQ ID NO 4 on ovine (o) GDF-9 and OGDF-9B stimulated 3H-thymidine incorporation of rat granulosa cells when added directly to the bioassay. Data presented are the mean and standard error of the mean of 4 replicate wells from one pool of granulosa cells; and
Figure 8 shows the effect of differing doses of a polyclonal antibody from sheep immunized against ovine GDF-9B peptide SEQ ID NO 5 on ovine (o) GDF-9 and oGDF-9B stimulated 3H-thymidine incorporation of rat granulosa cells when added directly to the bioassay. Data presented are the mean and standard error of the mean of 4 replicate wells from one pool of granulosa cells.
DETAILED DESCRIPTION OF THE INVENTION
The present invention provides a novel domain of functional significance at the N-terminal end of GDF-9 and GDF-9B. It is postulated that stimulation or inhibition of this domain by agonists or antagonists that interact therewith will be effective in modulating the ovulation rate of a female mammal.
The sequences of the N-terminal domains were compared with the corresponding sequences of the GDF-9 and GDF-9B proteins in a number of different species and a consensus sequence determined as shown in Figures 3 a and 3b for GDF-9 and GDF-9B respectively.
A number of peptides were then synthesised which corresponded with sequences within the N-terminal domains and which were anticipated to have an agonistic or antagonistic effect on the biological activity of GDF-9 and/or GDF-9B when administered in vivo. In particular, peptides were designed to be on the outside of the molecule according to its three dimensional structure; in a flexible region of the molecule; at least nine amino acids in length; non homologous with

other TGF beta family members; non convergent with other known proteins; in areas that did not contain a glycosylation site; and so that they could be coupled to a carrier protein. It was considered that the combination of these factors would result in peptides that would be highly specific across species and would not have cross-reactivity problems.
Thus, in a first embodiment, the present invention is directed to an isolated fragment of the GDF-9 N-terminal domain, having the ability to modulate ovulation in a female mammal, wherein said GDF-9 N-terminal domain consists of the amino acid sequence DQESASSELKKPLVPASVNLSEYFKQFLFPQNEC (SEQ ID NO: 1). The amino acid symbols correspond to the one letter code as set out in Table 1, below.
Preferably, the isolated peptide fragment comprises at least one amino acid for positions 1 to 9 or 25 to 34 of SEQ ID NO: 1.
Preferably the peptide comprises at least 5 contiguous amino acids of SEQ ID NO: 1, or a functional derivative, homolog, analog or mimetic thereof. More preferably, the peptide comprises at least 8, at least 10, at least 12, at least 14, at least 16, at least 18 or at least 20 contiguous amino acids of SEQ ID NO: 1, or a functional derivative, homolog, analog or mimetic thereof.
Most preferably the peptides is selected from the group comprising:
DQESASSELKKPLV(C) (SEQ ID NO: 3); and
SEYFKQFLFPQNEC (SEQ ID NO: 4);
or a functional derivative, homolog, analog or mimetic thereof. Amino acids in parentheses refer to amino acids optionally added for conjugation purposes.
The invention is also directed to an antibody or antibody fragment that binds to one or more GDF-9 peptides of the invention.
In a second embodiment the present invention is directed to an isolated peptide fragment of the GDF-9B N-terminal domain, having the ability to modulate ovulation in a female mammal, wherein said GDF-9B N-terminal domain consists of the amino acid sequence:

QAGSIASEVPGPSREHDGPESNQC (SEQ ID NO: 2), or a functional derivative, homology analog or mimetic thereof. The amino acid symbols correspond to the one letter code as set out in Table 1 below.
Preferably, the isolated peptide fragment comprises at least one amino acid from positions 1 to 5 or 21 to 23 of SEQ ID NO: 2.
Preferably, the peptide comprises at least 5 contiguous amino acids of SEQ ID NO: 2 or a functional derivative, analog, homolog or mimetic thereof. More preferably, the peptide comprises at least 8, at least 10, at least 12, at least 14, at least 16, at least 18 or at least 20 contiguous amino acids of SEQ ID NO: 2, or a functional derivative, homolog, analog or mimetic thereof.
Most preferably, the peptide is selected from the group comprising:
QAGSIASEVPGPSR(C) (SEQ ID NO: 5); and
SREHDGPESNQC (SEQ ID NO: 6);
or a functional derivative, homolog, analog or mimetic thereof.
The invention is also directed to an antibody or antibody fragment that binds to one or more GDF-9B peptides of the invention.
Amino acids in parentheses refer to amino acids optionally added for conjugation purposes.
The peptides of the present invention may be synthesised using known technology.
Analogs, derivatives or variants of the peptides of the invention may include sequence modifications or non-sequence modifications. Non-sequence modifications can include acetylation, methylation, phosphomethylation, carboxilation or glycosylation.
The specific N-tenninal peptides exemplified in the present invention are shown in relation to their position on the N-terminal portion of GDF-9 and GDF-9B in figures 1 and 2, respectively.
Preferred analogs include peptides who's sequence differs from those of the invention by one or more conservative amino acid substitutions, deletions or insertions which do not affect the

biological activity of the peptide. Conservative substitutions typically include the substitution of one amino acid for another with similar characteristics, e.g., substitutions within the following groups: valine, glycine; glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid; asparagine,- glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. Examples of conservative substitutions can also be found in the sequences of GDF-9 and GDF-9B in Figures 3 a and 3b whereby the substitutions in different mammalian species compared to the consensus sequence are shown. Other conservative substitutions can be taken from Table 1 below.
TABLE 1 CONSERVATIVE AMINO ACID REPLACEMENTS For Amino
Acid Code Replace with any of
Alanine A D-Ala, Gly, beta-Ala, L-Cys, D-Cys
Arginine R D-Arg, Lys, D-Lys, homo-Arg, D-homo-Arg,
Met, lie, D-Met, D-Ile, Orn, D-Orn
Asparagine N D-Asn, Asp, D-Asp, Glu, D-Glu, Gin, D-Gln
Aspartic Acid D D-Asp, D-Asn, Asn, Glu, D-Glu, Gin, D-Gln
Cysteine C D-Cys, S-Me-Cys, Met, D-Met, Thr, D-Thr
Glutamine Q D-Gln, Asn, D-Asn, Glu, D-Glu, Asp, D-Asp
Glutamic Acid E D-Glu, D-Asp, Asp, Asn, D-Asn, Gin, D-Gln
Glycine G Ala, D-Ala, Pro, D-Pro, .beta.-Ala Acp
Histidine H Asp, D-Asp, Lys, D-Lys, Tyr
Isoleucine I D-He, Val, D-Val, Leu, D-Leu, Met, D-Met
Leucine L D-Leu, Val, D-Val, Leu, D-Leu, Met, D-Met
Lysine K D-Lys, Arg, D-Arg, homo-Arg, D-homo-Arg,
Met, D-Met, He, D-Ile, Orn, D-Orn
Methionine M D-Met, S-Me-Cys, lie, D-He, Leu, D-Leu, Val,
D-Val
Phenylalanine F D-Phe, Tyr, D-Thr, L-Dopa, His, D-His, Trp,
D-Trp, Trans-3,4, or 5-phenylproline, cis-3,4, or
5-phenylproline
Proline P D-Pro, L-I-thioazolidine-4-carboxylic acid, D-
or L-l-oxazolidine-4-carboxylic acid

Serine S D-Ser, Thr, D-Thr, allo-Thr, Met, D-Met,
Met(0), D-Met(0), L-Cys, D-Cys
Threonine T D-Thr, Ser, D-Ser, allo-Tbr, Met, D-Met, Met(0),
D-Met(0), Val, D-Val
Tyrosine Y D-Tyr, Phe, D-Phe, L-Dopa, His, D-His
Valine V D-Val, Leu, D-Leu, lie, D-Ile, Met, D-Met
Other analogs include peptides with modifications which influence peptide stability. Such analogs may contain, for example, one or more non-peptide bonds (which replace the peptide bonds) in the peptide sequence. Also included are analogs that include residues other than naturally occurring L-amino acids, e.g. D-amino acids or non-naturally occurring synthetic amino acids, e.g. beta or gamma amino acids and cyclic analogs.
In a further aspect, the invention provides a use of the agonists and antagonists of the invention, in a method of modulating the ovulation rate of a female mammal, including human and non-human mammals. Such non-human mammals include sheep, cattle, goats, deer, pigs, horses, camelids, possums, non-human primates such as marmosets, cats, dogs and other commercially important species.
The method may comprise administering to said mammal an effective amount of one or more of said agonists or antagonists or a functional variant thereof, or an antibody or antibody fragment that binds thereto.
It is contemplated that equivalent N-terminal domain peptides of GDF-9 and GDF-9B of a particular mammal to be treated will be used in the methods of the present invention. Examples of GDF-9 and GDF-9B peptides from different species, and which correspond to the specific N-terminal peptides of SEQ ID NOs 3-6, are as shown in Table la, below.



The amino acid alignment of mature GDF-9 and GDF-9B of a number of species is shown in figures 3 a and 3b. These alignment sequences were generated with the multiple alignment programme of Vector NTL This programme uses the Clustal W algorithm (Thompson et al., 1994). The region encoding the mature region of the protein was identified and the nucleotide sequence was used to generate the predicted protein sequence. The sequences (GenBank accession number) used for alignment were as follows. GDF-9: cow (NM_174681), goat (AH014112), sheep (AF078545), pig (NM_001001909), dog (XM_538624), cat (in house data base), possum (AY033826)5 rabbit (in house data base), mouse (NM_008110), rat (NM_021672), chimpanzee (XM-27008) and human (NM_005260). GDF-9B: cow (AY572412), deer (in house data base), dog (XM_549005), cat (in house data base), possum (AH012378), chimpanzee (XM-29247), human (AF082350), mouse (NM_021670), rabbit (in house data base), pig (AF458070), goat (in house data base) and sheep (AF236079).
The modulation of the ovulation rate may comprise an increase or decrease in the ovulation rate of the female mammal by the administration of an agonist or antagonist of the invention to said animal resulting in antibodies being raised in vivo to said agonist or antagonist which in turn bind to the N-terminal domain of GDF-9 and/or GDF-9B to affect the biological activity thereof.

Without being bound by theory, it is considered that binding of the N-tenninal domain of GDF-9 and/or GDF-9B by an agonist/antagonist, and in particular by one or more antibodies raised against one or more peptides selected from the group comprising SEQ ID NOs: 3-6, results in altered circulating concentration of biologically active GDF-9 and/or GDF-9B. Where such a decrease comprises an approximate 50% fall in GDF-9B activity, an increase in ovulation rate has been observed previously, in particular, in Hanna Sheep where a single point mutation in the GDF-9B gene should result in half the amount of active GDF-9B in heterozygous animals and resulting in an increased ovulation rate and twinning. In homozygous animals, where there is no or very little circulating active GDF-9B, the animals were sterile (Galloway et al; 2000). Similarly, point mutations in the GDF-9 gene and concomitant modulation in ovulation rates have been observed in sheep (WO 03/102199; Hanrahan et ah, 2003).
Thus, it is postulated that an agonist or antagonist of the invention that results in a decrease of approximately 50% in the circulating levels of active GDF-9 and/or GDF-9B will result in an increase in ovulation rate, whilst an agonist or antagonist that results in a reduction in the circulation concentration of active GDF-9 and/or GDF-9B to approximately zero, will result in a decrease in ovulation and sterilisation in a female mammal.
Preferably the agonist or antagonist of the invention is an antibody which binds to the consensus N-terminal domains of GDF-9 and/or GDF-9B of SEQ ID NOs: 1 and 2. It should be appreciated that the term "antibody" encompasses fragments or analogues of antibodies which retain the ability to bind to a consensus binding domain defined herein, including but not limited to Fv, Fc, F(ab)2 fragments, ScFv molecules and the like. The antibody may be polyclonal or monoclonal, but is preferably monoclonal Such antibodies may be prepared by any technique known in the art for example (Juengel et a/., 2002) for administration to an animal, i.e. for use in passive immunisation. Alternatively, such antibodies may be produced in vivo by administration of an antigen in a suitable adjuvant, i.e. for use in active immunisation. Suitable adjuvants include Freund's complete or incomplete adjuvant, or similar immunostimulatory agent. The antibodies of the present invention may also be produced by genetic engineering methods such as chimeric and humanised monoclonal antibodies, comprising both human and non-human portions, which can be made using standard recombinant DNA techniques.

The present invention further contemplates the use of one or more agonists and/or antagonists of the present invention in combination with one or more active ingredients known to modulate ovulation rate to enhance the effect on ovulation. The active ingredients may be selected from the group comprising GDF-9; GDF-9B; BMPRII; BMPIB receptor (ALK6); ALK5 and BMP6; or functional fragments or variants thereof. In particular, the invention contemplates the use of BMPIB receptor in combination with an antibody or antibody fragment (Fc) that binds to a peptide of SEQ ID NOs: 5 or 6, in the modulation of ovulation in a female mammal.
The present invention further provides a pharmaceutical composition comprising at least one agonist or antagonist of the present invention together with a pharmaceutically acceptable carrier useful for the modulation of ovulation rate.
It is contemplated that the agonists or antagonists of the invention will be tested for biological activity in an animal model or in an in vitro model and suitably active compounds formulated into pharmaceutical compositions. The pharmaceutical compositions of the present invention may comprise, in addition to one or more agonists or antagonists of the present invention, a pharmaceutically acceptable excipient, carrier, buffer, stabiliser or other material well known in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient. The precise nature of the carrier or other material will be dependent upon the desired nature of the pharmaceutical composition, and the route of administration e.g. oral, intravenous, cutaneous, subcutaneous, intradermal, intramuscular or intraperitoneal.
Pharmaceutical compositions for oral administration may be in tablet, lozenge, capsule, powder, granule or liquid form. A tablet or other solid oral dosage form will usually include a solid carrier such as gelatine, starch, mannitol, crystalline cellulose, or other inert materials generally used in pharmaceutical manufacture. Similarly, liquid pharmaceutical compositions such as a syrup or emulsion, will generally include a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil or synthetic oil.
For intravenous, cutaneous, subcutaneous, intradermal or intraperitoneal injection, the active ingredient will be in the form of a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability.

In a further embodiment, the invention contemplates the use of one or more additional modulators of ovulation to be co-administered with the pharmaceutical composition of the present invention to give an additive or synergistic effect to the treatment regime. Examples of such additional modulators of ovulation include follicle stimulating hormone, Androvax (an androsteindione protein vaccine conjugate), and steroid hormone. Such modulators may be administered either separately, sequentially or simultaneously with at least one agonist or antagonist of the present invention depending upon whether ovulation is to be increased or decreased as will be appreciated by a skilled worker.
Administration of the pharmaceutical composition of the invention is preferably in a "therapeutically effective amount", this being sufficient to show the desired benefit to the individual. The actual amount administered, and rate and time-course of administration, will depend on the nature and severity of the female mammals underlying condition. Prescription of treatment, e.g. decisions on dosage etc., is within the responsibility of general practitioners and other medical doctors, and typically takes account of the disorder to be treated, the condition of the individual patient, the site of delivery, the method of administration and other factors known to practitioners. Examples of the techniques and protocols mentioned above can be found in Remington's Pharmaceutical Sciences, 16th edition, Oslo, A. (ed), 1980.
The invention will now be described in greater detail by reference to specific Examples, which should not be construed as in any way limiting the scope of the invention.
EXAMPLES
Example 1
Use of peptides and antibodies to the N-terminal domains of GDF-9 and GDF-9B to manipulate ovulation in ovine.
Four 12 to 15 mer peptides were synthesised corresponding to the N-terminal domains of the GDF-9 and GDF-9B protein sequences and including, where necessary, additional residues to facilitate conjugation to Keyhole Limpet Haemocyanin (KLH) to generate an antigen. The peptide sequences were:

DQESASSELKKPLV(C) (SEQ ID NO: 3);
SEYFKQFLFPQNEC (SEQ ID NO: 4);
QAGSIASEVPGPSR (C) (SEQ ID NO: 5); and
SREHDGPESNQC (SEQ ID NO: 6);
In this study, groups of 10 anoestrous Romney ewes were injected with 0.4 mg/ewe of each peptide-KLH conjugate antigen in Freunds complete adjuvant and 10 anoestrous Romney ewes were injected with 0.4 mg/ewe KLH antigen as a control group. Subsequently at monthly intervals on 4 occasions, the animals were boosted with further antigen (0.2 mg/ewe on each occasion) in a Span/Tween/Oil mixture (sc) and oestrous activity monitored using vasectomised rams during the breeding season. The ovulation rate was assessed by laparoscopy over 4 successive oestrous cycles and again at the termination of the experiment approximately 20-30 days later. The mean ovulation rate was determined by averaging these 5 observations for each ewe.
All of the control ewes displayed cyclical oestrous activity. The results of the effect of peptides of SEQ ID NOs: 3-6 on ovulation rate show a shutdown of ovulation rate in the peptide treated ewes compared to the control ewes which correlated with a significant increase in the levels of anti-GDF-9 and anti-GDF-9B serum antibodies as shown below in Table 2. Antibody levels were measured using an ELISA procedure after sheep plasma was diluted 1:20,000 as previously described (Juengel et al, 2002).
Peptides of SEQ ED NOs: 3-5 resulted in shutdown of ovulation rate in the majority of animals within 2 weeks after the first booster injection. The shutdown of ovulation rate was maintained until the termination of the experiment The peptide of SEQ ID NO:6 did not achieve significant shutdown of ovulation rate until the third observation (after 3 booster injections) and only achieved shutdown in 50% of animals. However, by the last booster injection, nine out of ten animals showed a shutdown in ovulation rate (Table 3). Shutdown of ovulation was measured by observation of the ovary whereby the ovary lacked externally visible corpora lutea. Ewes that were deemed to be in oestrus as assessed by observation of the uterus were excluded from the results of Table 3.


These data show unequivocally that a significant decrease in ovulation rate can be induced by the administration of a peptide antigen targeted to the N-terminal domain of GDF-9 or GDF-9B. In this particular example, the effect was caused by active immunisation (i.e. administration of an antigen to raise antibodies in vivo). However, it is predicted that a similar effect will be seen by passive immunisation, i.e. by administration of the antibodies per se.
Example 2
Use of peptides and antibodies to the N-terminal domains of GDF-9 and GDF-9B to manipulate ovulation in bovine.
Two 15-mer peptides were synthesised corresponding to the N-terminal domains of the bovine GDF-9 and GDF-9B protein sequence and including, wherein necessary, additional residues to facilitate conjugation to KLH to generate an antigen. The peptide sequences were:

DQESVSSELKKPLV(C) (SEQ ID NO: 7); and
QAGSIASEVPGPSR(C) (SEQ ID NO: 5).
Note that SEQ ID NO: 7 is a functional variant of SEQ ID NO: 3, comprising a single amino acid change (A-> V at amino acid position 5).
In this study, groups of 10 Friesian-cross heifers were injected with 0.4 mg/heifer of each peptide-KLH conjugate antigen in 2 ml Freund's complete adjuvant given as four 0.5 ml injections; 10 heifers were injected with 0.4 mg/heifer of both peptide-KLH conjugate antigens and 10 heifers were injected with KLH antigen as a control group. Subsequently at monthly intervals on 2 occasions, the animals were boosted with further antigen (0.2 mg/heifer on each occasion) in a Span/Tween/Marcol mixture (SC). Animals were slaughtered approximately two weeks after the second booster immunisation and the ovaries collected. Ovarian activity was determined by visual examination of the ovary. Ovulation rate was assigned based on the number of corpora lutea visible on the surface of the ovary. Corpora lutea were determined to be regressed (and thus not counted in ovulation rate score) if it appeared non functional by morphological criteria (small, lacking vascularization, pale colour) with an additional functional corpora lutea present or was non functional by morphological (small, lacking vascularization, pale colour) and endocrinological criteria (low levels of progesterone in blood samples collect at time of ovarian collection and 2 weeks previously). Follicular development was assessed on 5μm histological sections stained with hematoxylin and eosin.
Peripheral venous blood samples were collected (10 ml by heparinised vaccutainer) at the times of primary and first and second booster and also at 12 days after the first booster and again just before slaughter. The plasma samples were analyzed for antibody titres and progesterone concentrations.
All heifers appeared to have normal ovarian activity prior to immunization. Treatment with peptide of SEQ ID NO: 5, either alone or together with peptide of SEQ ID NO: 7 caused a significant alteration of ovarian activity as measured by ovulation rate. Treatment with peptide of SEQ ID NO: 7 alone showed twin ovulations of 30%, which is abnormally high, although this was not statistically significant with the number of animals examined. It is interesting to note that treatment with peptide of SEQ ID NO: 5 alone resulted in three


At collection, ovaries from heifers immunized with GDF-9B peptide (SEQ ID NO: 5) conjugated to KLH, GDF-9 peptide (SEQ ID NO 7) conjugated to KLH or the combination of GDF-9 peptide (SEQ ID NO: 7) conjugated to KLH and GDF-9B peptide (SEQ ID NO: 5) conjugated to KLH were noted to be morphologically different from the ovaries from the KLH immunized control heifers. The GDF-9 and/or GDF-9B immunized heifers had ovaries with fewer surface visible antral follicles. Similar observations were made when comparing histological sections of the ovaries (see Figure 4). This observation was quantified on 5 urn histological sections stained with hemotoxylin and eosin whereby the percentage of the total area of the section occupied by antral space, the number of antral follicles per unit area and the average size (area) of the follicle was measured (see Table 5, below). Treatment with peptide of SEQ ID NO 5, SEQ ID NO 7 or both, decreased (P<0.01) the total area of the section occupied by antral space, the number of antral follicles per unit area and the average size of the follicle (see Table 5, below). It thus appears from these results that the ovulation rate would decrease if immunisation with the peptide fragments of the invention were to be continued for a longer term. Indeed, some animals treated with peptide of SEQ ID NO: 5 (GDF-9B), demonstrated an increased incidence of ovarian shutdown compared to control (see Table 4, above).

TABLE5
Effects of immunization against keyhole limpet haemocyanin (KLH), growth differentiation factor 9 (GDF9; SEQ ID NO 7: DQESVSSELKKPLV(C)) peptide conjugated to KLH, growth differentiation fector 9B (GDF-9B, SEQ ID NO 5; QAGSIASEVPGPSR(C)) conjugated to KLH and GDF-9 peptide conjugated to KLH + GDF-9B peptide conjugated to KLH on the percentage of the tissue section area occupied by antral space, the total number of follicles corrected for area and the average follicle area.

These results demonstrate that the N-terminal peptides are active in modulating ovulation and follicular development in bovine. The dose given was the same as that for sheep, however in bovine the overall result was an increase in ovulation rather than a decrease. This is likely due to the "sheep dose" resulting in a weak immunisation resulting in reduced circulating level of active GDF-9 and/or GDF-9B, of approximately 50% which, as discussed above, results in an increase in ovulation rate.
This was confirmed by antibody titre data via ELISA as previously described (Juengel et al 2002) with minor modifications, see Table 6, below. The antigen coated onto the microtitre

plates was 200 ng ovine GDF-9 mature protein or 100 ng ovine GDF-9B mature protein. Antibodies were detected using a 1:20,000 dilution of a rabbit anti-bovine IgG. Sera from heifers was diluted to 1:500. This is a 40 times less dilution than was required for sheep sera in experiment 1, above, suggesting that the dose of GDF-9 and/or GDF-9B peptides given to bovine was a cweak' dose.
However, as mentioned above, it is likely that, given the decrease in follicular development, a decrease in ovulation rate may be induced in bovine by continuing the immunisation regime over a longer period.


It is expected that a larger dose in bovine, i.e. sufficient to reduce circulation levels of active GDF-9 and/or GDF-9B to approximately zero, or immunisation for a longer time period, will significantly reduce the ovulation rate in bovine.
These data show unequivocally that a significant modulation of ovulation rate and antral follicle development can be induced by the administration of a peptide antigen targeted at the N-terminal domain of GDF-9 and/or GDF-9B, indicating that the N-terminal mature regions of GDF-9 and GDF-9B are important for biological activity in catde. In this particular example, the effect was caused by active immunisation (i.e. administration of an antigen to raise antibodies in vivo). However, it is predicted that a similar effect will be seen by passive immunisation i.e. by administration of antibodies per se.
Example 3
Effect of antibodies raised against GDF-9 and GDF-9B peptide fragments on 3H-thymidine incorporation by rat granulosa cells in vitro.
Various antibody preparations were tested for their ability to neutralize the effects of ovine or murine GDF-9 and ovine GDF-9B on rat granulosa cells when added directly to the granulosa cell culture using a previously described method (McNatty et aL, 2005). A total of 100^g/ml of IgG was added for each treatment which was comprised of IgG purified from sheep immunised with the GDF-9 or GDF-9B peptide fragments with the balance of IgG purified for sheep immunised with KLH. The antibodies were able to neutralize the effects of the growth factor that they were directed against (see Figures 5 and 6). Furthermore, this response was shown to be dose dependent in the two antibody samples tested against ovine GDF-9 and GDF-9B (PO.001, see Figure 7 and 8).
Neutralization of the effects of ovine and murine GDF-9 and ovine GDF-9B by antibodies raised against peptide fragments of the N-terminal region of the protein (e.g. SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5) on incorporation of thymidine uptake by rat granulosa cells, taken together with the effects of active immunization with these peptides in sheep (see example 1) and cattle (see example 2), indicates that this region of die protein is important for biological activity in multiple species. These results also indicate that the antibodies against

the N-terminal domain of the mature region of GDF-9 and GDF-9B are effective in passively neutralizing GDF-9 and GDF-9B.
It will be apparent to the person skilled in the art that while the invention has been described in some detail for the purposes of clarity and understanding, various modifications and alternatives to the embodiments and methods described herein may be made without departing from the scope of the invention as defined in the appended claims.
INDUSTRIAL APPLICATION
The present invention provides compositions and methods for modulating the ovulation rate and therefore fertility in female mammals including humans.

REFERENCES
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2. McGrath SA, Esquela AF, Lee SJ : Oocyte-specific expression of growth differentiation factor-9. Mol Endocrinol 9:131-136,1995.
3. Laitinen M, Vuojolainen K, Jaatinen R, Ketola I, Aaltonen J, Lehtonen E, Heikinheimo M, Ritvos O: A novel growth differentiation factor-9 (GDF-9) related factor is co-expressed with GDF-9 in mouse oocytes during folliculogenesis. Mech Dev 78:135-140,1998.
4. Dube JL, Wang P, Elvin J, Lyons KM, Celeste AJ, Matzuk MM: The bone morphogenetic protein 15 gene is X-linked and expressed in oocytes. Mol Endocrinol 12:1809-1817,1998.
5. Jaatinen, R., Laitinen, M.P., Vuojolainen, K., Aaltonen, J., Louhio, H., Heikinheimo, K, Lehtonen, E. and Ritvos, O: Localisation of growth differentiation factor-9 (Gdf-9) mRNA and protein in rat ovaries and cDNA cloning of rat GDF-9 and its novel homolog GDF-9B.Mol Cell Endocrinol 156: 189-193,1999.
6. Aaltonen J, Laitinen M, Vuojolainen K, Jaatinen R, Horelli-Kuitunen N, Seppa L, Louhio H, Tuuri T, Sjoberg J, Butzow R, Hovatta O, Dale Ls Ritvos O: Human growth differentiation factor- 9 (GDF-9) and its novel homolog GDF-9B are expressed during early folliculogenesis. J Clin Endocrinol Metab 84: 2744-2750,1999.
7. Bodensteiner, K.J., Clay, CM., Moeller, C.L. and Sawyer, H.R.: Molecular cloning of the ovine growth/differentiation factor-9 gene and expression of growth/differentiation factor-9 in ovine and bovine ovaries biology of Reproduction 60,381-386,1999.

8. Bodensteiner, K.L, McNatty, K.P., Clay, CM., Moeller, C.L. and Sawyer, H.R.: Expression of growth and differentiation factor-9 in the ovaries of fetal sheep homozygous or heterozygous for the Inverdale prolificacy gene {FecX1). Biology of Reproduction 62:1479-1485,2000.
9. Galloway SM, McNatty KP, Cambridge LM, Laitinen MPE, Juengel JL9 Jokiranta TS, McLaren RJ, Luiro Ks Dodds KG, Montgomery GW, Beattie AE, Davis GH, Ritvos O: Mutations in an oocyte-derived growth factor gene (BMP15) cause increased ovulation rate and infertility in a dosage-sensitive manner. Nature Genetics 25:279-283,2000.
10. Eckery, D.C., Whale, L.J., Lawrence, S.B., Wilde, KA., McNatty, K.P. & Juengel, J.L.: Expression of mRNA encoding growth differentiation factor 9 and bone morphogenetic protein 15 during follicular formation and growth in a marsupial, the brushtail possum (Trichasurus vulpecula). Molecular & Cellular Endocrinology; 2002.
11. Yan, C, Wang, P., DeMayo, J., DeMayo, F.J., Elvin, J., Carino, C, Prasad, S.V., Skinner, S.S., Dunbar, B.S., Dube, J.L., Celeste, AJ- and Matzuk, M.M: Synergistic roles of bone morphogenetic protein 15 and growth differentiation factor 9 in ovarian function. Molecular Endocrinology 15: 854-866, 2001.
12. Fitzpatrick, S.L., Sindon, D.M., Shughue, PJ., Lane M.V., Merchenthaler, IJ., and Frail, D.F.: Expression of growth differntiating factor-9 messenger ribnucleic acid in ovarian and non-ovarian rodent and human tissue. Endocrinology, 139 (5), 2577-2578, 1998.
13. Hanrahan, LP., Gregan, S.M., Mulsant, P., Mullen, M., Davis, G.M., Powell, R., Galloway, S.M.,: Mutations in the genes for oocyte-derived growth factor GDF-9 and BMP-15 are associated with both increased ovulation rate and sterility in Cambridge and Belcare sheep (Ovis aeries). Biol. Reprod, 2003.

14. Juengel, J.L., Hudson, N.L., Heath, D.L., Smith, P., Reader, K.L., Lawrence, S.B., O'Connell, A.R.S Laitinen, M.P., Cranfield, M., Groome, N.P., Ritvos, O., McNatty, K.P.,: Growth differentiation factor 9 and bone morphogenic protein 15 are essential for ovarian follicular development in sheep. Biol. Reprod. 67(b), 1777-1789, 2002.
15. Jeffery, L., Mottershead, D.G., Myllymaa, S.s Gilchrist, R., Groome, N.? and Ritvos, O.,: Grouping of conserved regions (GoCore): Better prediction of function from sequences. Poster ESHRE Campus April, 2003.

16. Thompson JD, Higgins DG, Gibson TJ. CLUSTAL W: Improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 1994 22:4673-4680.
17. McNatty, K.P., Juengel, J.L., Reader, K.L., Lun, S-, Myllymaa, S.s Lawrence, S.B., Western, A., Meerasahib, M.F., Mottershead, D.G., Groome, N.P., Ritvos, O., Laitinen, M.P.E. (2005). Bone morphogenetic protein 15 and growth differentiation factor 9 cooperate to regulate granulosa cell function. Reproduction 129:473-480.
All references are incorporated into this specification in their entirety by means of this reference.

WHAT WE CLAIM IS:
1. An isolated fragment of the GDF-9 N-terminal domain, having the ability to modulate ovulation in a female mammal, wherein said GDF-9 N-terminal domain consists of the amino acid sequence DQESASSELKKPLVPASVNLSEYFKQFLFPQNEC (SEQ ED NO: 1), or a functional derivative, homolog, analog or mimetic thereof.
2. An isolated peptide fragment as claimed in claim 1 comprising at least one amino acid from positions 1 to 9 or 25 to 34 of SEQ ID NO: 1.
3. An isolated peptide fragment of the GDF-9B N-terminal domain, having the ability to modulate ovulation in a female mammal, wherein said GDF-9B N-terminal domain consists of the amino acid sequence QAGSIASEVPGPSREHDGPESNQC (SEQ ED NO: 2), or a functional derivative, homolog, analog or mimetic thereof.
4. An isolated peptide fragment as claimed in claim 3 comprising at least one amino acid from positions 1 to 5 or 21 to 23 of SEQ ID NO: 2.
5. An isolated peptide fragment of the GDF-9 N-terminal domain, having the ability to modulate ovulation in a female mammal wherein said fragment comprises at least one amino acid for positions 1 to 9 or 25 to 34 of SEQ ED NO: 1, or a functional derivative, homolog, analog or mimetic thereof.
6. An isolated peptide fragment of the GDF-9B N-terminal domain, having the ability to modulate ovulation in a female mammal, wherein said fragment comprises at least one amino acid from positions 1 to 5 or 21 to 23 of SEQ ID NO: 2 or a functional derivative, homolog, analog or mimetic thereof.
7. An isolated peptide fragment as claimed in any one of claims 1 to 6, comprising at least 5 contiguous amino acids of SEQ ID NO: 1 or SEQ ID NO: 2, or a functional derivative, homolog, analog or mimetic thereof

8. An isolated peptide fragment as claimed in claim 7, comprising at least 8 contiguous
amino acids of SEQ ID NO: 1 or SEQ ID NO; 2, or a functional derivative,
homology analog or mimetic thereof.
9. An isolated peptide fragment as claimed in claim 7, comprising at least 10
contiguous amino acids of SEQ ID NO: 1 or SEQ ID NO: 2, or a functional derivative, homolog, analog or mimetic thereof.
10. An isolated peptide fragment as claimed in claim 7, comprising at least 12
contiguous amino acids of SEQ ID NO: 1 or SEQ ID NO: 2, or a functional derivative, homolog, analog or mimetic thereof.
11. An isolated peptide fragment as claimed in claim 7, comprising at least 14
contiguous amino acids of SEQ ID NO: 1 or SEQ ID NO: 2, or a functional derivative, homolog, analog or mimetic thereof.
12. An isolated peptide fragment as claimed in claim 7, comprising at least 16
contiguous amino acids of SEQ ID NO: 1 or SEQ ED NO: 2, or a functional derivative, homolog, analog or mimetic thereof.
13. An isolated peptide fragment as claimed in claim 7, comprising at least 18
contiguous amino acids of SEQ ID NO: 1 or SEQ ID NO: 2, or a functional derivative, homolog, analog or mimetic thereof.
14. An isolated peptide fragment as claimed in claim 79 comprising at least 20
contiguous amino acids of SEQ ID NO: 1 or SEQ ID NO: 2, or a functional derivative, homolog, analog or mimetic thereof.
15. An isolated peptide fragment as claimed in any one of claims 1 to 14, selected from
the group comprising:
DQESASSELKKPLV(C) (SEQ ID NO: 3);
SEYFKQFLFPQNEC (SEQ ID NO: 4);
QAGSIASEVPGPSR(C) (SEQ ID NO: 5); and
SREHDGPESNQC (SEQ ID NO: 6);

or a functional derivative, homolog, analog or mimetic thereof.
16. An antibody or antibody fragment which binds to one or more peptide fragments of
any one of claims 1 to 15.
17. A method of modulating the ovulation rate in a female mammal, said method
comprising the step of administering to said mammal an effective amount of one or more isolated peptide fragments as claimed in any one of claims 1-15.
18. A method of modulating the ovulation rate in a female mammal, said method
comprising the step of administering to said mammal an effective amount of one or more antibodies or antibody fragments of claim 16.
19. A method of modulating the ovulation rate in a female mammal, said method
comprising the step of administering to said mammal an effective amount of one or more isolated peptide fragments as claimed in any one of claims 1 to 15 together with one or more antibodies or antibody fragments as claimed in claim 16.
20. A method of modulating the ovulation rate in a female mammal, comprising
administering to said mammal an effective amount of one or more peptide fragments selected from SEQ ID NOs 3-6, or a functional derivative, homolog, analog or mimetic thereof, and/or an antibody or antibody fragment that binds thereto.
21. A use of one or more isolated peptide fragments as claimed in any one of claims 1 to
15, in the manufacture of a medicament for modulating the ovulation rate of a female mammal.
22. A use of one or more antibodies or antibody fragments as claimed in claim 16, in the
manufacture of a medicament for modulating the ovulation rate of a female mammal.
23. A use of a) one or more isolated peptide fragments as claimed in any one of claims 1-
15 and b) one or more antibodies or antibody fragments as claimed in claim 16, in the manufacture of a medicament for modulating ovulation in a female mammal, wherein

the medicament is formulated for separate, sequential or simultaneous administration of a) and b).
24. A use as claimed in any one of claims 21 to 23, wherein the isolated peptide fragment
is selected from the group comprising SEQ ID NOs 3-6 or a functional derivative, homolog, analog or mimetic thereof.
25. A pharmaceutical composition comprising one or more isolated peptide fragments as
claimed in any one of claims 1 to 15, together with a pharmaceutically acceptable carrier or excipient.
26. A pharmaceutical composition comprising one or more antibodies or antibody
fragments as claimed in claim 16, together with a pharmaceutically acceptable carrier or excipient
27. A pharmaceutical composition comprising one or more isolated peptide fragments as
claimed in any one of claims 1 to 15 and one or more antibodies or antibody fragments as claimed in claim 16, together with a pharmaceutically acceptable carrier or excipient
28. A pharmaceutical composition comprising one or more peptides selected from the
group comprising SEQ ID NOs 3-6 or a functional variant thereof, and/or an antibody or antibody fragment that binds thereto, together with a pharmaceutically acceptable carrier or excipient
29. A method of modulating the ovulation rate in a female mammal, comprising
administering to said mammal an effective amount of a composition as claimed in any one of claims 25 to 28, in combination with one or more compounds selected from the group comprising GDF-9, GDF-9B, BMPRII, BMPIB receptor (ALK6), ALK5 and BMP6 or a functional fragment or variant thereof.
30. A method of modulating the ovulation rate in a female mammal comprising
administering to said mammal an effective amount of an antibody or antibody

fragment (Fc) that binds to a peptide of any one of SEQ ED NOS: 3 to 6, in combination with BMP IB receptor (ALK6).
31. A method of modulating the ovulation rate in a female mammal comprising
administering to said mammal an effective amount of a composition as claimed in any one of claims 25-28 in combination with a modulator of ovulation selected from the group consisting of FSH, androvax and steroid hormone.
32. A use of a) a composition as claimed in any one of claims 25 to 28 and b) one or more
compounds selected from the group comprising GDF-9, GDF-9B, BMPRII, BMPIB receptor (ALK6), ALK5 and BMP6 or a functional fragment or variant thereof in the manufacture of a medicament for modulating the ovulation rate in a female mammal, wherein the medicament is formulated for separate, sequential or simultaneous administration of a) and b).
33. A use of a) an antibody or antibody fragment (Fc) that binds to a peptide of any one of
SEQ ID NOS: 3-6 and b) BMPIB receptor (ALK6) in the manufacture of a medicament for modulating the ovulation rate in a female mammal, wherein the medicament is formulated for separate, sequential or simultaneous administration of a)andb).
34. A use of a) a composition as claimed in any one of claims 25 to 28 and b) a modulator
of ovulation selected from the group consisting of FSH, androvax and steroid hormone, in the manufacture of a medicament for modulating the ovulation rate in a female mammal wherein the medicament is formulated for separate, sequential or simultaneous administration of a) and b).